Vibrio cholerae, the causative agent of the disease cholera, is an important human pathogen that is an ongoing public health problem in regions of many developing countries in Latin America, Asia, South America and Africa. In many bacteria including V. cholerae, cyclic dimeric guanosine monophosphate (c-diGMP) regulates motility, biofilm formation and virulence. We have a solid general understanding of the basic biochemistry of c-diGMP turnover and c-diGMP- dependent phenotypes, but many critical questions remain. These include how does c-diGMP mechanistically control motile to biofilm life style switch in V. cholerae, how do environmental signals control c-di-GMP levels and biofilm formation in V. cholerae, and what are the consequences of c-diGMP signaling in the intestinal and aquatic and phases of V. cholerae life cycle. We will address these questions by focusing on two aims. In Aim 1, we will determine how specific key c-diGMP signaling proteins act to control motility. We will also analyze c- diGMP production during biofilm development and elucidate how motility status affects c-diGMP levels. In Aim 2, we will determine molecular mechanisms of environmental regulation of c- diGMP levels and analyze impact of c-diGMP signaling proteins in V. cholerae aquatic and intestinal life cycles. Our studies will lead to a better understanding of c-diGMP signal transduction pathways that are critical for the life cycle of V. cholerae and may, therefore, lead to new methods of combating this pathogen, as well as other disease-causing microorganisms that use c-diGMP signaling. This work will also contribute to the basic understanding of the newly emerging, ubiquitous signal transduction network and, therefore, will also be relevant to understand similar regulatory systems in a broad range of bacteria.